Photochromic materials, such as fluorescent proteins, can be used in a variety of applications and have particular utility as a tool in molecular biology (see, e.g., Irie, M. & Mori, M. J. Org. Chem. 53:803 (1988); Parthenopoulos, D. A. & Rentzepis, P. M. Science 245:843 (1989); Hanazawa, M., et al. J. Chem. Soc. Chem. Commun. 206 (1992); Dvornikov, A. S., et al. J. Phys. Chem. 98:6746-6752 (1994); Dvornikov, A. S. & Rentzepis, P. M. Opt. Mem. Neur. Netw. 3:75-86 (1994); U.S. Pat. No. 4,471,470; and U.S. Pat. No. 5,325,324). However, the number of known fluorescent proteins is limited.
In general, photochromic materials have two states that can be interconverted by irradiation (see, e.g., U.S. Pat. No. 6,046,925). For example, the fluorescence behavior of wild-type (WT) green fluorescent protein (GFP) is known to have two absorption maxima, one at 395 nm, the other at 465 nm, but only one emission peak at 490 nm, indicating a common excited state (see, e.g., Heim, R., et al. Proc. Nat. Acad. Sci., USA 91:12501 (1994)). These absorption peaks have been attributed to the neutral and anionic fluorophore states, respectively, which can be interconverted by proton transfer between the fluorophore and Glu222 (see, e.g., Cubitt, A. B., et al. Trends in Biochem. Sci. 20:448 (1995); Chattoraj, M., et al. Proc. Nat. Acad. Sci., USA 93, 8362 (1996); and Brejc, K., et al. Proc. Nat. Acad. Sci., USA 94:2306-2311 (1997)). Ser65 and Thr203 are particularly close to the chromophore in GFPs (see, e.g., Ormo, M., et al. Science 273:1392 (1996); Yang, F., et al. Nature Biotech. 14:1246 (1996)). Consequently, these residues can influence the photophysical properties of the protein. Alteration of Ser65 strongly favors ionization of the chromophore by hindering solvation and ionization of Glu222, whereas mutational loss of the Thr203 hydroxyl exerts a weaker opposing effect by reducing the solvation of the anionic form (see, e.g., Ormo, M., et al. Science 273:1392 (1996); Yang, F., et al. Nature Biotech. 14:1246 (1996). Aromatic residues at position 203 of GFP increase the peak excitation wavelength by 13-24 nm, probably by increasing the polarizability around the chromophore through p—p interactions (see, e.g., Ormo, M., et al. Science 273:1392 (1996)).
The fluorescent proteins of various aquatic organisms, such as those in the phylum Cnidaria, act as energy-transfer acceptors in bioluminescence and thereby emit fluorescence (see, e.g., Ward, W. W., et al., Photochem. Photobiol., 35:803-808 (1982); and Levine, L. D., et al., Comp. Biochem. Physiol., 72B:77-85 (1982). GFP proteins have been modified to alter their excitation and emission spectra. Specifically, a variety of GFPs have been constructed by modifying the amino acid sequence of a naturally-occurring (or wild-type) GFP from Aequorea Victoria (see, e.g., Prasher, D. C., et al., Gene, 111:229-233 (1992); Heim, R., et al., Proc. Natl. Acad. Sci., USA, 91:12501-04 (1994); U.S. Ser. No. 08/337,915, filed Nov. 10, 1994; International application PCT/US95/14692, filed Nov. 10, 1995; and U.S. Ser. No. 08/706,408, filed Aug. 30, 1996. Further, the cDNA of GFP can be concatenated with those encoding other proteins; and the resulting fusion polypeptides can be fluorescent and retain the biochemical features of the partner proteins (see, e.g., Cubitt, A. B., et al., Trends in Biochem. Sci. 20:448-455 (1995)). Mutagenesis studies have produced GFP mutants with shifted wavelengths of excitation or emission (see, e.g., Heim, R. & Tsien, R. Y. Current Biol. 6:178-182 (1996); and Tsien, R. Y., et al., Trends Cell Biol. 3:242-245 (1993).
In addition, mutations in Aequorea fluorescent proteins, referred to as “folding mutations,” improve the ability of fluorescent proteins to fold at higher temperatures and to be more fluorescent when expressed in mammalian cells, but have little or no effect on the peak wavelengths of excitation and emission. Such mutations can be combined with mutations that influence the spectral properties of GFP to produce proteins with altered spectral and folding properties. In addition, new fluorescent proteins based on GFP have been identified by random screening of GFPs (see, for example, Heim, R., et al. Proc. Natl. Acad. Sci. USA 91:12501-12504 (1994); Ehrig, et al. FEBS Lett. 367:163-166 (1995); and Delagrave, et al. Bio/Technology 13:151-154 (1995)).
The utility of fluorescent proteins as a tool in molecular biology has prompted the search for other proteinaceous fluorophores with different and improved properties, as compared to known fluorescent proteins. Thus, there is a need for the isolation and characterization of new fluorescent proteins that exhibit properties not currently available in the limited number of known fluorescent proteins.
Accordingly, an object of the present invention is to provide novel fluorescent proteins for use as a tool in molecular biology. In particular, an object of the present invention is to provide methods and compositions comprising a PFP, including for example a recombinant PFP and, more particularly, a fusion PFP for use in in vitro and in vivo biological assays, including screening assays and cellular assays.